Eggs and ingredients derived from eggs are important food commodities; they are used in a vast number of different food formulations. Eggs contain a number of different proteins. Many of these proteins contribute to the excellent functional properties that eggs can invoke in a food system. Egg yolk contains an array of complex proteins, most of which are complexed with carbohydrate or lipid groups (Nakamura and Doi, 2000). Egg white (egg albumen) contains up to 40 different proteins, many of which play very important roles in the functional attributes of egg white (Kilara and Harwalkar, 1996).
One of the most practical roles albumen proteins play is to form foams when an egg albumen solution is exposed to gas supersaturation or mechanical forces (Walstra, 1996; Nakamura and Doi, 2000). Commercial products of egg white (liquid, frozen, and dehydrated egg white) are processed according to strict specifications to provide optimum foaming properties. To achieve superior foaming ability, yolk contamination and heat damage (from drying or pasteurization) should be minimized (Froning, 1994). The quality of egg foam is greatly dependent on the protein concentration and the environmental conditions under which the foam is formed (such as pH and ionic strength) (Hammershoj and Qvist, 2001). High quality foams are typically produced at pH's of 4–5 and 8–9. The way the foam is made is also an important factor that can affect the properties of the foam (Kilara and Harwalkar, 1996). To complicate matters further, the quality of the foam is also a function of the initial quality of the egg albumen (e.g. ratio of firm vs. thin albumen), storage conditions of the eggs, age of the eggs, and the source (different hen lines) of the eggs (Hammershoj and Qvist, 2001). These factors contribute to the great variability seen with egg albumen foams. These factors also make it difficult to compare results reported throughout the literature.
Foams are colloidal systems in which tiny air bubbles are dispersed in an aqueous continuous phase (Damodaran, 1997). The general mechanism behind foam formation is that upon experiencing a force (mechanical whipping or shaking) or supersaturation of a gas, the egg proteins come in contact with the air-water interface and are partly adsorbed to it and, at the same time, partly denature at the interface. This happens because it becomes more favorable for the protein to expose its hydrophobic interior to the air phase, while leaving its hydrophilic part in contact with solution. The proteins form a film at the air-water interface, and this film traps air to form bubbles (Nakamura and Doi, 2000), collectively making a foam. This foam is stabilized by a variety of forces, including the viscosity of the liquid phase and electrostatic and steric forces of the proteins making up the air bubbles (Walstra, 1996). A variety of destabilizing forces also concurrently tend to minimize the foam formation and break down the foam. These include electrostatic attractions (and strong repulsion in very charged proteins) and hydrophobic attraction between the proteins (Walstra, 1996).
For egg albumen, it has been found that different proteins have markedly different foaming properties (Nakamura, 1963; Johnson and Zabik, 1981). These different properties are a result of differences in the structures of these proteins. The prior art is limited regarding the role the different egg albumen proteins play in foamability and foam stability under different conditions. Even less has been reported regarding their effects on the physical properties of the foams (e.g., viscoelasticity and surface properties).
It is generally accepted that egg albumen proteins that form good foams are those that adsorb rapidly and readily denature at the air surface (Nakamura and Doi, 2000). These proteins are capable of reducing surface tension of the air-water interface readily, which is the main factor behind good foam formation. On the other hand, proteins that adsorb slowly and that do not readily unfold are those that contribute to good foam stability (but not good foamability); these proteins form an ordered and stable network at their air interface (Graham and Philips, 1976).
There have been some efforts to connect protein conformational changes with changes in the ability of the proteins to form foams. In summary, it has been found that foaming properties are affected primarily by (a) surface hydrophobicity of the protein (more hydrophobicity=more foaming ability), (b) protein charge density and charge distribution (increased charge leads to too much repulsion and poor foam stability), and (c) protein flexibility (more flexible proteins form foams more readily) (Damodaran, 1997). These physiochemical properties of the proteins are highly influenced by the nature of the environment in which they exist.
Some attempts have been made to increase the foaming functionality of some food proteins. Research on modifying the structure of food proteins to improve foaming properties has focused mainly on heat denaturation experiments. Zhu and Damodaran (1994) showed that heat-induced partial unfolding of whey proteins, as compared to extensive unfolding, increased the foamability and foam stability of the protein. Heat denaturation has in some cases been shown to increase the foaming properties of egg albumen proteins (Kilara and Harwalkar, 1996). This increase can be attributed to an increase in the surface hydrophobicity of the proteins, primarily ovalbumin (Damodaran, 1997; Nakamura and Doi, 2000). However, if heat treatment is too extensive, protein concentration too high, or the protein is at a pH and ionic strength that favors aggregation, coagulation may occur, which adversely affects foaming properties (Kilara and Harwalkar, 1996; Oakenfull et al. 1997).
Until relatively recently, it was a common view that proteins followed a two state behavior upon unfolding—going directly from the native to a fully unfolded random coil state (Dill, 1990). Much information has arisen in the last two decades that points to a far more complex situation than that for many proteins. Some proteins go through stable intermediate states on unfolding, and others unfold only partially, depending on specific solvent conditions (such as specific pH, ionic strength, and ion type). Altering the pH of a protein medium is one method that can be used to unfold proteins. All proteins contain basic and acidic amino acids, resulting in a net charge on the protein. The net charge of the protein is increased as the pH travels in either direction from the protein isoelectric point up to a point where the charge repulsion causes the protein molecule to unfold (Creighton, 1993). Proteins unfold at extremes of pH because the unfolded configuration becomes more energetically favorable under these conditions (Dill and Shortle, 1991). Under these conditions, the hydrophobic forces, which normally account for most of protein stability, are not strong enough to counteract the electrostatic free energy brought about at low or high pH.
Protein unfolding (i.e., denaturation) can also be accomplished by changing the solvent conditions to disrupt the forces that stabilize the protein. This also brings about conditions favoring the extended configuration of the protein. Depending on the functional property desired for the protein, this can have a detrimental or a beneficial effect on proteins.
The extent of unfolding and the configuration a protein molecule possesses at extreme pH values has been found to be very protein-dependent (Goto et al., 1990a; Goto and Fink, 1990). Numerous reports, mainly at low pH, suggest that many proteins may only be partly unfolded under extreme pH conditions while others are extensively unfolded (e.g., Goto and Fink, 1989; Goto et al., 1990a,b; Fink et al., 1994; Nishii et al., 1995). Similar observations have been made, for some proteins, when ionic strength is increased at low pH (i.e., as salts are added) (Goto and Fink, 1994; Fink et al., 1994). Different intermediate states at low pH can be explained by differences in contribution from charge repulsion, which acts to expand and unfold the protein, and hydrophobic forces, which work to contract the structure and stabilize it (Dill and Shortle, 1991; Goto and Nishikiori, 1991). Further decreases in pH or addition of salts can cause partial refolding due to charge screening effects or direct binding of the added anions; this leads to a partial collapse of the extended protein chain (as electrostatic repulsion between the charged unfolded protein chain is lowered; hydrophobic interactions would strengthen in this situation). These intermediate structures, brought about by extreme pH or a combination of pH and salts, are referred to as “molten globules” as they still retain some characteristics of the native state while in other aspects closely resemble the fully unfolded state. Just as with the native state, the properties of this ensemble of non-native states depend very sensitively on the solution conditions.
The molten globular state of proteins has been largely or completely ignored in food science research, with only a few workers recently realizing the potential and role of partially unfolded proteins in food systems (Hirose, 1993; Matsumara et al., 1994; Dickinson and Matsumara, 1994; Tatsumi et al., 1999). Conformational changes at highly alkaline pH have likewise been given little consideration, but results for one enzyme (β-lactamase) indicate that very high pH can lead to a partial unfolding of this protein due to the presence of cations from the bases, just as the presence of anions do at low pH (Goto and Fink, 1989).
Many food proteins have characteristics that allow them to have excellent functionality for gelation, emulsification, foaming, and the like. Studies indicate that after treating some of these proteins to obtain these functionalities, the proteins take on non-native conformations (Dickinson and Matsumura, 1994). Several proteins commonly found in or used in food products have been found to take on a state characteristic of the molten globule under mild denaturing conditions, including extremes of pH. These include myoglobin (Goto et al., 1990a,b), ovotransferrrin (Hirose and Yamashita, 1991), serum albumen (Lee and Hirose, 1992), alpha-lactoglobulin (Dickinson and Matsumura, 1994), ovalbumen (Tatsumi et al., 1999), hemoglobin, and myosin (Kristinsson, 2002a, b; Kristinsson and Hultin, 2003a, b). This has not heretofore been investigated for egg albumen proteins.
Direct evidence for the molten globule state in food systems is difficult to achieve as the proteins are either aggregated (gelation) or adsorbed to another phase (emulsification and foaming). However, indirect evidence is available. α-lactalbumen, a protein found in milk, has been extensively studied and has been found to be more readily absorbed to an oil-water interface in its molten globular state (Dickinson and Matsumura, 1994; Matsumura et al., 1994). Cytochrome c in its molten globular state at pH 3.5 was also found to more readily adsorb to an interface of air and water (Gazova et al., 1999). The molten globule is characterized by a non-specific assembly of secondary structure segments, loss of tertiary interactions, and partial exposure of hydrophobic clusters to solvent (Kuwajima, 1989).
Kitabatake and Doi (1987) reported that foamed ovalbumen has increased reactive —SH groups, a situation also observed in the molten globule state of this protein at acid pH (Tatsumi et al., 1999). Recent studies have suggested that in order for a protein to travel across or be inserted into hydrophobic cell membranes, it takes on a configuration characteristic of the molten globule (Sedlak and Antalik, 1998; Gazova et al., 1999).
Studies on the molten globules discussed above have been done at extremes of pH (pH 1–3 and pH 11–13), which are not practical pH values for most food systems, including those that would employ egg white.
There has been no known study, even suggested or proposed, regarding how controlled acid and alkali denaturation, followed by pH readjustment to renaturing conditions, affects the foaming properties or conformation of the albumen proteins, collectively or individually.